Previous studies have shown that cholinergic neurons can induce acetylcholine receptor (AChR) clusters. Our studies indicate that other cell surface remodeling changes occur at the early stages of synapse formation. These changes include the neuronal growth cone filopodial insertion into the coated regions of the post synaptic plasma membrane and the appearance of coated pits and vesicles, some of which contain Alpha-bungarotoxin (AlphaBTX) binding sites. In this investigation we will determine how coated pits and vesicles contribute to pre and postsynaptic membrane remodeling and AChR aggregation during early stages of synapse formation. For this purpose nerve-muscle co-cultures will constitute the model of nerve-muscle interactions. Ciliary ganglia will be the source of cholinergic neurons, and dorsal root ganglia the noncholinergic neurons. Because the growth cone is likely to influence the specific interactions between the neuron and the postsynaptic cell, we have designed studies to investigate membrane organization and coated vesicle distribution in four readily identifiable stages defined by the relationship of the growth cone to the postsynaptic cell. These studies will determine whether there are specific domains in the plasma membrane at which receptors cluster, which coated pits and vesicles contain AChR's, whether these are exocytic or endocytic, and which vesicles share common determinants with the Golgi cisternae and/or with the plasma membrane. Our work will employ double label experiments utilizing a monoclonal antibody (mAb35) which binds to the AChRs Alpha subunit, immunogold procedures and cytochemical markers for the Golgi cisternae, as well as relocation techniques to coordinate ultrastructural and immunocytochemical studies allowing us to section the same neuronal growth cone-muscle contact observed by light microscopy. The appearance of Alpha-BTX-TMR stained AChR clusters will be monitored throughout the developmental course using time-lapse and silicon intensification photography. Analysis of AChR density using 125IAlpha-BTX and autoradiography will permit us to quantitate pools of pre-existing AChRs versus newly inserted AChRs at each stage. Such quantitative data on AChR localization will be correlated with the information obtained on the distribution of coated pits and vesicles. These studies will allow us to determine how coated pits and vesicles may contribute to the surface membrane remodeling and the accumulation of AChRs at the synapse.